PROJECT SUMMARY Human airway diseases such as cystic fibrosis (CF), asthma and COPD are becoming increasingly prevalent and there is a need to develop more effective therapies. CF is an airway disease that affects 1 in 3,000 live births in the U.S. Respiratory failure is the most common cause of death. Despite intense efforts to target CFTR, the gene that causes CF, only recently have there been clinically efficacious therapies2. This discrepancy highlights the necessity to better understand the molecular mechanisms of CF and to develop better methods to test candidate drugs. One of the major roadblocks in airway disease research is the lack of ex vivo models that accurately reflect airway tissue. Traditional culture systems and animal models of airway disease have limitations with regard to clinical relevance, as they do not recapitulate human pathobiology. Current in vitro airway models typically consist of an epithelial monolayer cultured on filter inserts. This model is an incomplete representation of the in vivo tissue environment. The absence of natural extracellular matrix, and the inability to co-culture cells in a three-dimensional arrangement, prohibit the assessment of more complex cell-cell interactions. The lung-on-a-chip (LOAC) systems offer the possibility of overcoming some of these limitations. However, materials employed to mold microfluidic devices are extremely stiff and can alter epithelial cell function. Therefore, data from transwell cultures or LOAC are not an accurate reflection of disease or therapeutic responses to drugs, which may lead to an increased likelihood of failure at later stages in drug development. In this application, we will develop an airway model that better mimics human airway characteristic features and physiology by incorporating iPSC-derived cells and a natural scaffold into a bioreactor system. This novel culture system will be an invaluable tool to model airway disease and potential therapies. First, this technology makes it possible to use CF patient-specific stem cells for mechanistic studies, and to identify new treatments. Moreover, and maybe more importantly, this approach will study CF patient-specific cells in a more physiologically relevant model that recapitulates in vivo conditions better than existing culture systems. Two aims are proposed to develop an ex vivo airway model to study CF disease that provides a unique method to screen for potential therapies: 1) We will engineer an airway model from human basal cells and airway fibroblasts from F508del CF patients in a bioreactor system, and we will evaluate the capacity of our model to replicate airway epithelial cell function and the characteristic features of delF508CF disease. 2) We will make a similar system using human iPSCs-basal cells from delF508 CF patient. To take advantage of this CF iPSCs model, we will study the effect of Pseudomonas and viral infection, on exaggerated inflammation, alterations in ion transport, and mucus hypersecretion in delF508 CF. We plan to evaluate the impact of two known CFTR correctors, VX-770 and VX-809, on modulating the function of the CFTR protein in our system.